Abstract

Alkaline conformers of cytochrome c may be involved in both its electron transport and apoptotic functions. We use cobalt(II)bis(terpyridine), Co(terpy)2(2+), as a reagent for conformationally gated electron-transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochrome c expressed in Escherichia coli , WT*K79H, with alanine at position 72 and Saccharomyces cerevisiae , yK79H, with trimethyllysine (Tml) at position 72. Co(terpy)2(2+) is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co(terpy)2(2+) occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79-heme alkaline conformer directly reduced by Co(terpy)2(2+). The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72?Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition.

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